Engineered primate cystine/cysteine degrading enzymes as antineogenic agents

ABSTRACT

Methods and compositions related to the engineering of a protein with L-cyst(e)ine degrading enzyme activity are described. For example, in certain aspects there may be disclosed a modified cystathionine-γ-lyase comprising one or more amino acid substitutions and capable of degrading L-cyst(e)ine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with L-cyst(e)ine using the disclosed proteins or nucleic acids.

The present application claims the priority benefit of U.S. provisionalapplication Nos. 61/871,727, filed Aug. 29, 2013 and 61/948,106, filedMar. 5, 2014, the entire contents of which are incorporated herein byreference.

The invention was made with government support under Grant No. R01CA154754 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of medicine andbiology. More particularly, it concerns compositions and methods for thetreatment of cancer with enzymes that deplete both L-cystine andL-cysteine. Even more particularly, it concerns the engineering of aprimate enzyme with high cysteine/cysteine degrading activity andstability suitable for human therapy.

2. Description of Related Art

Systemic depletion of various amino acids has been shown to be effectivein killing a wide variety of tumor types with minimal toxicity tonon-cancerous tissues. This therapeutic effect can be achieved throughthe use of pharmacologically optimized enzymes introduced intocirculation that degrade the amino acid upon which the tumor relies.Certain cancers, such as prostate, small cell lung carcinomas,glioblastomas, and hepatocellular carcinomas, have been shown to beheavily dependent on extracellular cysteine/cystine in order toproliferate and survive. Many of these tumors aberrantly overexpress thexCT(−) cystine/glutamate antiporter in order to maintain sufficientcysteine levels needed for protein and glutathione production,suggesting that they have lost or down-regulated their native cysteinebiosynthetic capacity. In support of this idea, the use of smallmolecule inhibitors of xCT(−) cystine/glutamate antiporters, such assulfasalazine, have been shown to retard the growth of prostate andsmall cell lung cancer tumor xenografts (Doxsee et al., 2007; Guan etal., 2009). Although a blockade of the xCT(−) dependent transport ofL-cystine is promising it does not eliminate the transport of freeL-cysteine by the Na⁺-dependent ASC transporter and/or Na⁺-independenttransporters, and in some examples free L-cysteine is provided to tumorcells by bone-marrow derived stromal cells (Zhang et al., 2012). Atherapeutic that depletes both cystine and cysteine can thus completelydeprive tumors of this essential metabolite. However, there are no knownirreversible cysteine/cystine degrading enzymes with properties oractivity suitable to be applied as a human therapeutic, and in general,enzymes frequently do not exist that can accomplish specific amino aciddegradation, necessitating the engineering of desired activities fromexisting enzymes.

SUMMARY OF THE INVENTION

The present invention concerns the engineering of primatecystathionine-gamma-lyase (CGL) enzymes such that both L-cystine andL-cysteine (referred to herein as L-cyst(e)ine) can be efficientlydegraded from serum, and providing the modified CGL enzymes in aformulation suitable for human cancer therapy. To develop an enzymedisplaying low K_(M) and high catalytic activity, k_(cat), as comparedto the native enzyme, the inventors engineered the native enzyme bymodifying selected amino acids, which modifications result in an enzymehaving dramatically improved enzymatic properties. As such, CGL enzymesmodified as described herein overcome a major deficiency in the art byproviding novel enzymes that comprise human or primate polypeptidesequences having L-cyst(e)ine-degrading catalytic activity as comparedto the native enzyme. As such, these modified enzymes may be suitablefor cancer therapy and have low immunogenicity and improved serumstability.

Accordingly, in a first embodiment there is provided a modifiedpolypeptide, particularly an enzyme variant with L-cyst(e)ine degradingactivity derived from primate enzymes related to cystathionine-γ-lyase(CGL) enzymes. For example, a novel enzyme variant may have an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2-6. Forexample, the variant may be derived from a human enzyme, such as humanCGL. In certain aspects, there may be a polypeptide comprising amodified primate CGL capable of degrading L-cyst(e)ine. In someembodiments, the polypeptide may be capable of degrading L-cyst(e)ineunder physiological conditions. For example, the polypeptide may have acatalytic efficiency for L-cyst(e)ine (k_(cat)/K_(M)) of at least orabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000,10⁴, 10⁵, 10⁶ s⁻¹M⁻¹ or any range derivable therein.

An unmodified polypeptide may be a native CGL, particularly a humanisoform or other primate isoform. For example, the native human CGL mayhave the sequence of SEQ ID NO: 1. Non-limiting examples of other nativeprimate CGL include Pongo abelii CGL (Genbank ID: NP_001124635.1; SEQ IDNO: 7), Macaca fascicularis CGL (Genbank ID: AAW71993.1; SEQ ID NO: 8),Pan troglodytes CGL (Genbank ID: XP_513486.2; SEQ ID NO: 9), and Panpaniscus CGL (Genbank ID: XP_003830652.1; SEQ ID NO: 10). Exemplarynative polypeptides include a sequence having about, at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity (orany range derivable therein) to SEQ ID NOs: 1 or 7-10 or a fragmentthereof. For example, the native polypeptide may comprise at least or upto about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,350, 400, 405 residues (or any range derivable therein) of the sequenceof SEQ ID NOs: 1 or 7-10.

In some embodiments, the native CGL may be modified by one or more othermodifications, such as chemical modifications, substitutions,insertions, deletions, and/or truncations. In a particular embodiment,the native CGL may be modified by substitutions. For example, the numberof substitutions may be one, two, three, four or more. In furtherembodiments, the native CGL may be modified in the substrate recognitionsite or any location that may affect substrate specificity. For example,the modified polypeptide may have the at least one amino acidsubstitution at an amino acid position corresponding to amino acidposition 59 and/or 339 of SEQ ID NOs: 1 or 7-10. In these examples, thefirst methionine of each sequence corresponds to amino acid position 1,and each amino acid is numbered sequentially therefrom.

In certain embodiments, the substitutions at amino acid positions 59and/or 339 are threonine (T) or valine (V). In particular embodiments,the modification are one or more substitutions selected from the groupconsisting of 59T and 339V. In a further embodiment, the substitutionsmay comprise the 59T substitution. In a still further embodiment, thesubstitutions may comprise an additional substitution of 339V.

In some embodiments, the native CGL may be a human CGL. In a particularembodiment, the substitutions are a combination of E59T and E339V ofhuman CGL (for example, the modified polypeptide having the amino acidsequence of SEQ ID NO: 2, a fragment or homolog thereof). In furtherembodiments, the modified polypeptide may be a Pongo abelii CGL-TVmutant (SEQ ID NO: 3), Macaca fascicularis CGL-TV mutant (SEQ ID NO: 4),Pan troglodytes CGL-TV mutant (SEQ ID NO: 5), or Pan paniscus CGL-TVmutant (SEQ ID NO: 6).

A modified polypeptide as discussed above may be characterized as havinga certain percentage of identity as compared to an unmodifiedpolypeptide (e.g., a native polypeptide) or to any polypeptide sequencedisclosed herein. For example, the unmodified polypeptide may compriseat least or up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 405 residues (or any range derivable therein)of a native primate CGL (i.e., human, Pongo abelii, Macaca fascicularis,Pan troglogytes, or Pan paniscus CGL). The percentage identity may beabout, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% (or any range derivable therein) between the unmodifiedportions of a modified polypeptide (i.e., the sequence of the modifiedpolypeptide excluding any substitutions at amino acids 59 and/or 339)and the corresponding native polypeptide. It is also contemplated thatpercentage of identity discussed above may relate to a particularmodified region of a polypeptide as compared to an unmodified region ofa polypeptide. For instance, a polypeptide may contain a modified ormutant substrate recognition site of CGL that can be characterized basedon the identity of the amino acid sequence of the modified or mutantsubstrate recognition site of CGL to that of an unmodified or mutant CGLfrom the same species or across species. For example, a modified ormutant human polypeptide characterized as having at least 90% identityto an unmodified CGL means that at least 90% of the amino acids in thatmodified or mutant human polypeptide are identical to the amino acids inthe unmodified polypeptide.

In some aspects, the present invention also contemplates polypeptidescomprising the modified CGL linked to a heterologous amino acidsequence. For example, the modified CGL may be linked to theheterologous amino acid sequence as a fusion protein. In a particularembodiment, the modified CGL may be linked to amino acid sequences, suchas an IgG Fc, albumin, an albumin binding peptide, or an XTENpolypeptide for increasing the in vivo half-life.

To increase serum stability, the modified CGL may be linked to one ormore polyether molecules. In a particular embodiment, the polyether maybe polyethylene glycol (PEG). The modified polypeptide may be linked toPEG via specific amino acid residues, such as lysine or cysteine. Fortherapeutic administration, such a polypeptide comprising the modifiedCGL may be dispersed in a pharmaceutically acceptable carrier.

In some aspects, a nucleic acid encoding such a modified CGL iscontemplated. In one aspect, the nucleic acid has been codon optimizedfor expression in bacteria. In particular embodiments, the bacteria isE. coli. In other aspects, the nucleic acid has been codon optimized forexpression in a fungus (e.g., yeast), in insect cells, or in mammaliancells. The present invention further contemplates vectors, such asexpression vectors, containing such nucleic acids. In particularembodiments, the nucleic acid encoding the modified CGL is operablylinked to a promoter, including but not limited to heterologouspromoters. In one embodiment, a modified CGL may be delivered to atarget cell by a vector (e.g., a gene therapy vector). Such viruses mayhave been modified by recombinant DNA technology to enable theexpression of the modified CGL-encoding nucleic acid in the target cell.These vectors may be derived from vectors of non-viral (e.g., plasmids)or viral (e.g., adenovirus, adeno-associated virus, retrovirus,lentivirus, herpes virus, or vaccinia virus) origin. Non-viral vectorsare preferably complexed with agents to facilitate the entry of the DNAacross the cellular membrane. Examples of such non-viral vectorcomplexes include the formulation with polycationic agents whichfacilitate the condensation of the DNA and lipid-based delivery systems.An example of a lipid-based delivery system would include liposome baseddelivery of nucleic acids.

In still further aspects, the present invention further contemplateshost cells comprising such vectors. The host cells may be bacteria(e.g., E. coli), fungal cells (e.g., yeast), insect cells, or mammaliancells.

In some embodiments, the vectors are introduced into host cells forexpressing the modified CGL. The proteins may be expressed in anysuitable manner. In one embodiment, the proteins are expressed in a hostcell such that the protein is glycosylated. In another embodiment, theproteins are expressed in a host cell such that the protein isaglycosylated.

In some embodiments, the polypeptides or nucleic acids are in apharmaceutical formulation comprising a pharmaceutically acceptablecarrier. The polypeptide may be a native primate CGL polypeptide or amodified CGL polypeptide. The nucleic acid may encode a native primateCGL polypeptide or a modified CGL polypeptide.

Certain aspects of the present invention also contemplate methods oftreatment by the administration of the native primate CGL peptide, thenucleic acid encoding the native primate CGL peptide in a gene therapyvector, the modified CGL peptide, the nucleic acid encoding the modifiedCGL in a gene therapy vector, or the formulation of the presentinvention, and in particular methods of treating tumor cells or subjectswith cancer. The subject may be any animal, such as a mouse. Forexample, the subject may be a mammal, particularly a primate, and moreparticularly a human patient. In some embodiments, the method maycomprise selecting a patient with cancer. In certain aspects, thesubject or patient may be maintained on a L-cyst(e)ine-restricted dietor a normal diet.

In some embodiments, the cancer is any cancer that is sensitive toL-cyst(e)ine depletion. In one embodiment, the present inventioncontemplates a method of treating a tumor cell or a cancer patientcomprising administering a formulation comprising such a polypeptide. Insome embodiments, the administration occurs under conditions such thatat least a portion of the cells of the cancer are killed. In anotherembodiment, the formulation comprises such a modified CGL withL-cyst(e)ine degrading activity at physiological conditions and furthercomprising an attached polyethylene glycol chain. In some embodiment,the formulation is a pharmaceutical formulation comprising any of theabove discussed CGL variants and pharmaceutically acceptable excipients.Such pharmaceutically acceptable excipients are well known to those ofskill in the art. All of the above CGL variants may be contemplated asuseful for human therapy.

In a further embodiment, there may also be provided a method of treatinga tumor cell comprising administering a formulation comprising anon-bacterial (mammalian, e.g., primate or mouse) modified CGL that hasL-cyst(e)ine degrading activity or a nucleic acid encoding thereof.

Because tumor cells are dependent upon their nutrient medium forL-cyst(e)ine, the administration or treatment may be directed to thenutrient source for the cells, and not necessarily the cells themselves.Therefore, in an in vivo application, treating a tumor cell includescontacting the nutrient medium for a population of tumor cells with theengineered (i.e., modified) CGL. In this embodiment, the medium can beblood, lymphatic fluid, spinal fluid and the like bodily fluid whereL-cyst(e)ine depletion is desired.

In accordance with certain aspects of the present invention, such aformulation containing the modified CGL can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intrasynovially, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularly, orally,topically, by inhalation, infusion, continuous infusion, localizedperfusion, via a catheter, via a lavage, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art.

In a further embodiment, the method may also comprise administering atleast a second anticancer therapy to the subject. The second anticancertherapy may be a surgical therapy, chemotherapy, radiation therapy,cryotherapy, hormone therapy, immunotherapy or cytokine therapy.

In one embodiment, a composition comprising a modified CGL or a nucleicacid encoding a modified CGL is provided for use in the treatment of atumor in a subject. In another embodiment, the use of a modified CGL ora nucleic acid encoding a modified CGL in the manufacture of amedicament for the treatment of a tumor is provided. Said modified CGLmay be any modified CGL of the embodiments.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding,” with reference to anucleic acid, are used to make the invention readily understandable bythe skilled artisan; however, these terms may be used interchangeablywith “comprise” or “comprising,” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Schematic of cyst(e)inease enzyme catalysis: hCGL and engineeredhCGL variants convert L-cystine to pyruvate, ammonia, and thiocysteine.Thiocysteine is non-enzymatically degraded to hydrogen sulfide andL-cysteine. L-cysteine is further degraded by hCGL and engineered hCGLvariants to pyruvate, ammonia, and hydrogen sulfide.

FIGS. 2A-2B—Michaelis-Menten kinetics of hCGL-TV catalyzed degradationof (A) L-cystine (open circle) and (B) L-cysteine (solid circle).

FIG. 3—Activity over time of hCGL-TV (open circle) in pooled human serumincubated at 37° C., with an apparent T_(0.5) of 228±6 h.

FIGS. 4A-4B—Prostate tumor lines (A) DU145 and (B) PC3 treated withvarying concentrations of hCGL-TV, with apparent IC₅₀ values ˜60 nM foreither cell line.

FIG. 5—Amino acid sequence of hCGL-TV (SEQ ID NO: 11). Contains anN-terminal His₆ tag and the hCGL amino acid sequence from residue 2-405with mutations E59T-E339V underlined in bold.

FIGS. 6A-6B—(A) A single 50 mg/kg i.p. dose of PEG-hCGL-TV in FVB mice(n=5/group) ablated cystine levels for over 96 h, and (B) loweredcysteine levels for over 48 h. (***p<0.001, **p<0.01, *p<0.05, andNS=Not significant.

FIG. 7—The pharmacokinetics from a single 50 mg/kg i.p. dose ofPEG-hCGL-TV in FVB mice (n=5/group) was followed over time resulting inan absorption T_(1/2) of ˜23 h, and an elimination T_(1/2) of 40±7 h.

FIG. 8—FVB mice bearing HMVP2 prostate carcinoma allografts (n=8 eachgroup) were treated i.p. with PEG-hCGL-TV at 50 mg/kg body weight (opensquare), PEG-hCGL-TV at 100 mg/kg body weight (solid triangle), PBS(open circle), or heat-inactivated PEG-hCGL-TV at 100 mg/kg body weight(solid diamond) once every 4 days.

FIG. 9—Male nude mice bearing human (PC3) prostate carcinoma xenografts(n=7 each group) were treated i.p. with PEG-hCGL-TV at 50 mg/kg bodyweight (solid triangle), PEG-hCGL-TV at 100 mg/kg body weight (solidcircle), PBS (solid diamond), or heat inactivated PEG-hCGL-TV at 100mg/kg body weight (solid square) once every 4 days.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Cysteine is considered a non-essential amino acid as it can besynthesized from homocysteine derived from the essential amino acidL-methionine via the transsulfuration pathway, which comprises theenzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CGL).Thus, the depletion of cysteine is expected to be relatively non-toxicto normal tissues with an intact transsulfuration pathway. Certaincancers may have abnormally low or absent expression of transsulfurationpathway enzymes CBS and/or CGL thus requiring them to importL-cysteine/L-cystine from the extracellular compartment. Inhepatocellular carcinomas, the downregulation of CBS was furthercorrelated with poor prognosis (Kim et al., 2009), and gastrointestinalcancers have also been observed to have frequent epigenetic silencing ofCBS (Zhao et al., 2012). In other examples, the absence of CGLexpression has been frequently observed in lymphoblastic leukemia celllines (Glode et al., 1981; Link et al., 1983). The expression levels ofCBS and CGL, as well as xCT(−) cysteine transporter, constituteimportant tumor biomarkers for patient selection for treatment with acysteine/cysteine depletion regimen.

The present invention provides engineered, therapeutic enzymes thatdegrade L-cyst(e)ine. Also provided are methods of using said enzymes totreat diseases, such as cancer, lysosomal storage disease (i.e.,cistinosis), and to abrogate adverse immune effects in a variety ofautoimmune conditions. Thus, a therapeutic enzyme that can deplete theseamino acids may have utility as an immune modulating agent.

I. DEFINITIONS

As used herein the terms “protein” and “polypeptide” refer to compoundscomprising amino acids joined via peptide bonds and are usedinterchangeably.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining proteins or protein fragments operably linked in a non-nativeway.

As used herein, the term “half-life” (½-life) refers to the time thatwould be required for the concentration of a polypeptide thereof to fallby half in vitro or in vivo, for example, after injection in a mammal.

The terms “in operable combination,” “in operable order,” and “operablylinked” refer to a linkage wherein the components so described are in arelationship permitting them to function in their intended manner, forexample, a linkage of nucleic acid sequences in such a manner that anucleic acid molecule capable of directing the transcription of a givengene and/or the synthesis of desired protein molecule, or a linkage ofamino acid sequences in such a manner so that a fusion protein isproduced.

The term “linker” is meant to refer to a compound or moiety that acts asa molecular bridge to operably link two different molecules, wherein oneportion of the linker is operably linked to a first molecule, andwherein another portion of the linker is operably linked to a secondmolecule.

The term “PEGylated” refers to conjugation with polyethylene glycol(PEG), which has been widely used as a drug carrier, given its highdegree of biocompatibility and ease of modification. PEG can be coupled(e.g., covalently linked) to active agents through the hydroxy groups atthe end of the PEG chain via chemical methods; however, PEG itself islimited to at most two active agents per molecule. In a differentapproach, copolymers of PEG and amino acids have been explored as novelbiomaterial that would retain the biocompatibility of PEG, but thatwould have the added advantage of numerous attachment points permolecule (thus providing greater drug loading), and that can besynthetically designed to suit a variety of applications.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full-lengthcoding sequence or by any portion of the coding sequence so as thedesired enzymatic activity is retained.

The term “native” refers to the typical form of a gene, a gene product,or a characteristic of that gene or gene product when isolated from anaturally occurring source. A native form is that which is mostfrequently observed in a natural population and is thus arbitrarilydesignated the normal or wild-type form. In contrast, the term“modified,” “variant,” or “mutant” refers to a gene or gene product thatdisplays modification in sequence and functional properties (i.e.,altered characteristics) when compared to the native gene or geneproduct.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for an RNA capable of beingtranscribed. In some cases, RNA molecules are then translated into aprotein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic composition (such as a therapeuticpolynucleotide and/or therapeutic polypeptide) that is employed inmethods to achieve a therapeutic effect. The term “therapeutic benefit”or “therapeutically effective” as used throughout this applicationrefers to anything that promotes or enhances the well-being of thesubject with respect to the medical treatment of this condition. Thisincludes, but is not limited to, a reduction in the frequency orseverity of the signs or symptoms of a disease. For example, treatmentof cancer may involve, for example, a reduction in the size of a tumor,a reduction in the invasiveness of a tumor, reduction in the growth rateof the cancer, or prevention of metastasis. Treatment of cancer may alsorefer to prolonging survival of a subject with cancer.

The term “K_(M)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction. The term “k_(cat)” as used hereinrefers to the turnover number or the number of substrate molecules eachenzyme site converts to product per unit time, and in which the enzymeis working at maximum efficiency. The term “k_(cat)/K_(M)” as usedherein is the specificity constant, which is a measure of howefficiently an enzyme converts a substrate into product.

The term “cystathionine-γ-lyase” (CGL or cystathionase) refers to anyenzyme that catalyzes the hydrolysis of cystathionine to cysteine. Forexample, it includes primate forms of cystathionine-γ-lyase, orparticularly, human forms of cystathionine-γ-lyase.

“Treatment” and “treating” refer to administration or application of atherapeutic agent to a subject or performance of a procedure or modalityon a subject for the purpose of obtaining a therapeutic benefit of adisease or health-related condition. For example, a treatment mayinclude administration of a pharmaceutically effective amount of acyst(e)inease.

“Subject” and “patient” refer to either a human or non-human, such asprimates, mammals, and vertebrates. In particular embodiments, thesubject is a human.

II. CYSTATHIONINE-γ-LYASE

A lyase is an enzyme that catalyzes the breaking of various chemicalbonds, often forming a new double bond or a new ring structure. Forexample, an enzyme that catalyzed this reaction would be a lyase:ATP→cAMP+PP_(i). Lyases differ from other enzymes in that they onlyrequire one substrate for the reaction in one direction, but twosubstrates for the reverse reaction.

A number of pyrioxal-5′-phosphate (PLP)-dependent enzymes are involvedin the metabolism of cysteine, homocysteine, and methionine, and theseenzymes form an evolutionary related family, designated as Cys/Metmetabolism PLP-dependent enzymes. These enzymes are proteins of about400 amino acids and the PLP group is attached to a lysine residuelocated in the central location of the polypeptide. Members of thisfamily include cystathionine-γ-lyase (CGL), cystathionine-γ-synthase(CGS), cystathionine-β-lyase (CBL), methionine-γ-lyase (MGL),O-acetylhomoserine (OAH)/O-acetyl-serine (OAS) sulfhydrylase (OSHS).Common to all of them is the formation of a Michaelis complex leading toan external substrate aldimine. The further course of the reaction isdetermined by the substrate specificity of the particular enzyme.

For example, the inventors introduced specific mutations into aPLP-dependent lyase family member, cystathionine-γ-lyase, to change itssubstrate specificity. In this manner the inventors produced novelvariants with the de novo ability to degrade both L-cystine andL-cysteine. In other embodiments, a modification of other PLP-dependentenzymes for producing novel L-cyst(e)ine degrading activity may also becontemplated.

Cystathionine-γ-lyase (CGL or cystathionase) is an enzyme which breaksdown cystathionine into cysteine and α-ketobutyrate. Pyridoxal phosphateis a prosthetic group of this enzyme. As shown in Examples, proteinengineering was used to convert cystathionase, which has only weakactivity for the degradation of L-cysteine and L-cystine, into an enzymethat can degrade these amino acids at a high rate.

III. CYST(E)INEASE ENGINEERING

Some embodiments concern modified proteins and polypeptides. Particularembodiments concern a modified protein or polypeptide that exhibits atleast one functional activity that is comparable to the unmodifiedversion, preferably, the L-cyst(e)ine degrading activity. In furtheraspects, the protein or polypeptide may be further modified to increaseserum stability. Thus, when the present application refers to thefunction or activity of “modified protein” or a “modified polypeptide,”one of ordinary skill in the art would understand that this includes,for example, a protein or polypeptide that possesses an additionaladvantage over the unmodified protein or polypeptide, such as theL-cyst(e)ine degrading activity. In certain embodiments, the unmodifiedprotein or polypeptide is a native CGL, preferably a human CGL. It isspecifically contemplated that embodiments concerning a “modifiedprotein” may be implemented with respect to a “modified polypeptide,”and vice versa.

Determination of activity may be achieved using assays familiar to thoseof skill in the art, particularly with respect to the protein'sactivity, and may include for comparison purposes, for example, the useof native and/or recombinant versions of either the modified orunmodified protein or polypeptide. For example, the L-cyst(e)inedegrading activity may be determined by any assay to detect theproduction of any substrates resulting from the degradation of L-cystineand/or L-cysteine, such as the detection of pyruvate using3-methyl-2-benzothiazolinone hydrazone (MBTH) (Takakura et al., 2004).

In certain embodiments, a modified polypeptide, such as a modified CGL,may be identified based on its increase in L-cyst(e)ine degradingactivity. For example, substrate recognition sites of the unmodifiedpolypeptide may be identified. This identification may be based onstructural analysis or homology analysis. A population of mutantsinvolving modifications of such substrate recognitions sites may begenerated. In a further embodiment, mutants with increased L-cyst(e)inedegrading activity may be selected from the mutant population. Selectionof desired mutants may include methods for the detection of byproductsor products from L-cyst(e)ine degradation.

Modified proteins may possess deletions and/or substitutions of aminoacids; thus, a protein with a deletion, a protein with a substitution,and a protein with a deletion and a substitution are modified proteins.In some embodiments, these modified proteins may further includeinsertions or added amino acids, such as with fusion proteins orproteins with linkers, for example. A “modified deleted protein” lacksone or more residues of the native protein, but may possess thespecificity and/or activity of the native protein. A “modified deletedprotein” may also have reduced immunogenicity or antigenicity. Anexample of a modified deleted protein is one that has an amino acidresidue deleted from at least one antigenic region that is, a region ofthe protein determined to be antigenic in a particular organism, such asthe type of organism that may be administered the modified protein.

Substitution or replacement variants typically contain the exchange ofone amino acid for another at one or more sites within the protein andmay be designed to modulate one or more properties of the polypeptide,particularly its effector functions and/or bioavailability.Substitutions may or may not be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine, ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

In addition to a deletion or substitution, a modified protein maypossess an insertion of residues, which typically involves the additionof at least one residue in the polypeptide. This may include theinsertion of a targeting peptide or polypeptide or simply a singleresidue. Terminal additions, called fusion proteins, are discussedbelow.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%, or between about 81% and about90%, or even between about 91% and about 99% of amino acids that areidentical or functionally equivalent to the amino acids of a controlpolypeptide are included, provided the biological activity of theprotein is maintained. A modified protein may be biologicallyfunctionally equivalent to its native counterpart in certain aspects.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

IV. ENZYMATIC L-CYST(E)INE DEGRADATION FOR THERAPY

In certain aspects, the polypeptides may be used for the treatment ofdiseases, including cancers that are sensitive to L-cyst(e)inedepletion, such as hepatocellular carcinoma, melanoma, and renal cellcarcinoma, with novel enzymes that deplete L-cystine and/or L-cysteine.The invention specifically discloses treatment methods using modifiedCGL with L-cyst(e)ine degrading activity. Certain embodiments of thepresent invention provide novel enzymes with L-cyst(e)ine degradingactivity for increased therapeutic efficacy.

Certain aspects of the present invention provide a modified CGL withL-cyst(e)ine degrading activity for treating diseases, such as tumors.In one example, the modified polypeptide may have human polypeptidesequences and thus may prevent allergic reactions in human patients,allow repeated dosing, and increase the therapeutic efficacy.

Tumors for which the present treatment methods are useful include anymalignant cell type, such as those found in a solid tumor or ahematological tumor. Exemplary solid tumors can include, but are notlimited to, a tumor of an organ selected from the group consisting ofpancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney,larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary hematological tumors include tumors of the bone marrow, T or Bcell malignancies, leukemias, lymphomas, blastomas, myelomas, and thelike. Further examples of cancers that may be treated using the methodsprovided herein include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer(including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer and gastrointestinal stromal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, varioustypes of head and neck cancer, melanoma, superficial spreading melanoma,lentigo malignant melanoma, acral lentiginous melanomas, nodularmelanomas, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairycell leukemia, multiple myeloma, acute myeloid leukemia (AML) andchronic myeloblastic leukemia.

The cancer may specifically be of the following histological type,though it is not limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; malignantmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

The engineered primate cyst(e)inease derived from CGL may be used hereinas an antitumor agent in a variety of modalities for depleting L-cystineand/or L-cysteine from a tumor cell, tumor tissue, or the circulation ofa mammal with cancer, or for depletion of L-cystine and/or L-cysteinewhere its depletion is considered desirable.

Depletion can be conducted in vivo in the circulation of a mammal, invitro in cases where L-cystine and/or L-cysteine depletion in tissueculture or other biological mediums is desired, and in ex vivoprocedures where biological fluids, cells, or tissues are manipulatedoutside the body and subsequently returned to the body of the patientmammal. Depletion of L-cystine and/or L-cysteine from circulation,culture media, biological fluids, or cells is conducted to reduce theamount of L-cystine and/or L-cysteine accessible to the material beingtreated, and therefore comprises contacting the material to be depletedwith a L-cystine- and/or L-cysteine-degrading amount of the engineeredprimate cyst(e)inease under L-cystine- and/or L-cysteine-degradingconditions as to degrade the ambient L-cystine and/or L-cysteine in thematerial being contacted.

Because tumor cells may be dependent upon their nutrient medium forL-cystine and/or L-cysteine, the depletion may be directed to thenutrient source for the cells, and not necessarily the cells themselves.Therefore, in an in vivo application, treating a tumor cell includescontacting the nutrient medium for a population of tumor cells with theengineered cyst(e)inease. In this embodiment, the medium may be blood,lymphatic fluid, spinal fluid and the like bodily fluid where L-cystineand/or L-cysteine depletion is desired.

L-cystine- and/or L-cysteine-degrading efficiency can vary widelydepending upon the application, and typically depends upon the amount ofL-cystine and/or L-cysteine present in the material, the desired rate ofdepletion, and the tolerance of the material for exposure tocyst(e)inease. L-cystine and/or L-cysteine levels in a material, andtherefore rates of L-cystine and/or L-cysteine depletion from thematerial, can readily be monitored by a variety of chemical andbiochemical methods well known in the art. Exemplary L-cystine- and/orL-cysteine-degrading amounts are described further herein, and can rangefrom 0.001 to 100 units (U) of engineered cyst(e)inease, preferablyabout 0.01 to 10 U, and more preferably about 0.1 to 5 U engineeredcyst(e)inease per milliliter (mL) of material to be treated.

L-cystine- and/or L-cysteine-degrading conditions are buffer andtemperature conditions compatible with the biological activity of a CGLenzyme, and include moderate temperature, salt, and pH conditionscompatible with the enzyme, for example, physiological conditions.Exemplary conditions include about 4-40° C., ionic strength equivalentto about 0.05 to 0.2 M NaCl, and a pH of about 5 to 9, whilephysiological conditions are included.

In a particular embodiment, the invention contemplates methods of usingengineered cyst(e)inease as an antitumor agent, and therefore comprisescontacting a population of tumor cells with a therapeutically effectiveamount of engineered cyst(e)inease for a time period sufficient toinhibit tumor cell growth.

In one embodiment, the contacting in vivo is accomplished byadministering, by intravenous or intraperitoneal injection, atherapeutically effective amount of a physiologically tolerablecomposition comprising an engineered cyst(e)inease of this invention toa patient, thereby depleting the circulating L-cystine and/or L-cysteinesource of the tumor cells present in the patient. The contacting ofengineered cyst(e)inease can also be accomplished by administering theengineered cyst(e)inease into the tissue containing the tumor cells.

A therapeutically effective amount of an engineered cyst(e)inease is apredetermined amount calculated to achieve the desired effect, i.e., todeplete L-cystine and/or L-cysteine in the tumor tissue or in apatient's circulation, and thereby cause the tumor cells to stopdividing. Thus, the dosage ranges for the administration of engineeredcyst(e)inease of the invention are those large enough to produce thedesired effect in which the symptoms of tumor cell division and cellcycling are reduced. The dosage should not be so large as to causeadverse side effects, such as hyperviscosity syndromes, pulmonary edema,congestive heart failure, and the like. Generally, the dosage will varywith age of, condition of, sex of, and extent of the disease in thepatient and can be determined by one of skill in the art. The dosage canbe adjusted by the individual physician in the event of anycomplication.

For example, a therapeutically effective amount of an engineeredcyst(e)inease may be an amount such that when administered in aphysiologically tolerable composition is sufficient to achieve aintravascular (plasma) or local concentration of from about 0.001 toabout 100 units (U) per mL, preferably above about 0.1 U, and morepreferably above 1 U engineered cyst(e)inease per mL. Typical dosagescan be administered based on body weight, and are in the range of about5-1000 U/kilogram (kg)/day, preferably about 5-100 U/kg/day, morepreferably about 10-50 U/kg/day, and more preferably about 20-40U/kg/day.

The engineered cyst(e)inease can be administered parenterally byinjection or by gradual infusion over time. The engineered cyst(e)ineasecan be administered intravenously, intraperitoneally, orally,intramuscularly, subcutaneously, intracavity, transdermally, dermally,can be delivered by peristaltic means, can be injected directly into thetissue containing the tumor cells, or can be administered by a pumpconnected to a catheter that may contain a potential biosensor orL-cyst(e)ine.

The therapeutic compositions containing engineered cyst(e)inease areconventionally administered intravenously, as by injection of a unitdose, for example. The term “unit dose” when used in reference to atherapeutic composition refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent, i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also contemplated and are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Exemplary multipleadministrations are described herein and are particularly preferred tomaintain continuously high serum and tissue levels of engineeredcyst(e)inease and conversely low serum and tissue levels ofL-cyst(e)ine. Alternatively, continuous intravenous infusion sufficientto maintain concentrations in the blood in the ranges specified for invivo therapies are contemplated.

V. CONJUGATES

Compositions and methods of the present invention involve engineeredcyst(e)ineases, such as by forming conjugates with heterologous peptidesegments or polymers, such as polyethylene glycol. In further aspects,the engineered cyst(e)ineases may be linked to PEG to increase thehydrodynamic radius of the enzyme and hence increase the serumpersistence. In certain aspects, the disclosed polypeptide may beconjugated to any targeting agent, such as a ligand having the abilityto specifically and stably bind to an external receptor or binding siteon a tumor cell (U.S. Patent Publ. 2009/0304666).

A. Fusion Proteins

Certain embodiments of the present invention concern fusion proteins.These molecules may have the modified cystathionase linked at the N- orC-terminus to a heterologous domain. For example, fusions may alsoemploy leader sequences from other species to permit the recombinantexpression of a protein in a heterologous host. Another useful fusionincludes the addition of a protein affinity tag, such as a serum albuminaffinity tag or six histidine residues, or an immunologically activedomain, such as an antibody epitope, preferably cleavable, to facilitatepurification of the fusion protein. Non-limiting affinity tags includepolyhistidine, chitin binding protein (CBP), maltose binding protein(MBP), and glutathione-S-transferase (GST).

In a particular embodiment, the cyst(e)inease may be linked to a peptidethat increases the in vivo half-life, such as an XTEN polypeptide(Schellenberger et al., 2009), IgG Fc domain, albumin, or albuminbinding peptide.

Methods of generating fusion proteins are well known to those of skillin the art. Such proteins can be produced, for example, by de novosynthesis of the complete fusion protein, or by attachment of the DNAsequence encoding the heterologous domain, followed by expression of theintact fusion protein.

Production of fusion proteins that recover the functional activities ofthe parent proteins may be facilitated by connecting genes with abridging DNA segment encoding a peptide linker that is spliced betweenthe polypeptides connected in tandem. The linker would be of sufficientlength to allow proper folding of the resulting fusion protein.

B. Linkers

In certain embodiments, the engineered cyst(e)inease may be chemicallyconjugated using bifunctional cross-linking reagents or fused at theprotein level with peptide linkers.

Bifunctional cross-linking reagents have been extensively used for avariety of purposes, including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies. Suitablepeptide linkers may also be used to link the engineered cyst(e)inease,such as Gly-Ser linkers.

Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino-,sulfhydryl-, guanidine-, indole-, carboxyl-specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis, and themild reaction conditions under which they can be applied.

A majority of heterobifunctional cross-linking reagents contain aprimary amine-reactive group and a thiol-reactive group. In anotherexample, heterobifunctional cross-linking reagents and methods of usingthe cross-linking reagents are described (U.S. Pat. No. 5,889,155,specifically incorporated herein by reference in its entirety). Thecross-linking reagents combine a nucleophilic hydrazide residue with anelectrophilic maleimide residue, allowing coupling, in one example, ofaldehydes to free thiols. The cross-linking reagent can be modified tocross-link various functional groups.

Additionally, any other linking/coupling agents and/or mechanisms knownto those of skill in the art may be used to combine primate engineeredcyst(e)inease, such as, for example, antibody-antigen interaction,avidin biotin linkages, amide linkages, ester linkages, thioesterlinkages, ether linkages, thioether linkages, phosphoester linkages,phosphoramide linkages, anhydride linkages, disulfide linkages, ionicand hydrophobic interactions, bispecific antibodies and antibodyfragments, or combinations thereof.

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andtherapeutic/preventative agents. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo.These linkers are thus one group of linking agents.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use ofsuch cross-linkers is well understood in the art. Another embodimentinvolves the use of flexible linkers.

Once chemically conjugated, the peptide generally will be purified toseparate the conjugate from unconjugated agents and from othercontaminants. A large number of purification techniques are availablefor use in providing conjugates of a sufficient degree of purity torender them clinically useful.

Purification methods based upon size separation, such as gel filtration,gel permeation, or high performance liquid chromatography, willgenerally be of most use. Other chromatographic techniques, such asBlue-Sepharose separation, may also be used. Conventional methods topurify the fusion proteins from inclusion bodies may be useful, such asusing weak detergents, such as sodium N-lauroyl-sarcosine (SLS).

C. PEGylation

In certain aspects of the invention, methods and compositions related toPEGylation of engineered cyst(e)inease are disclosed. For example, theengineered cyst(e)inease may be PEGylated in accordance with the methodsdisclosed herein.

PEGylation is the process of covalent attachment of poly(ethyleneglycol) polymer chains to another molecule, normally a drug ortherapeutic protein. PEGylation is routinely achieved by incubation of areactive derivative of PEG with the target macromolecule. The covalentattachment of PEG to a drug or therapeutic protein can “mask” the agentfrom the host's immune system (reduced immunogenicity and antigenicity)or increase the hydrodynamic size (size in solution) of the agent, whichprolongs its circulatory time by reducing renal clearance. PEGylationcan also provide water solubility to hydrophobic drugs and proteins.

The first step of the PEGylation is the suitable functionalization ofthe PEG polymer at one or both terminals. PEGs that are activated ateach terminus with the same reactive moiety are known as“homobifunctional,” whereas if the functional groups present aredifferent, then the PEG derivative is referred as “heterobifunctional”or “heterofunctional.” The chemically active or activated derivatives ofthe PEG polymer are prepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For proteins, typical reactive amino acidsinclude lysine, cysteine, histidine, arginine, aspartic acid, glutamicacid, serine, threonine, and tyrosine. The N-terminal amino group andthe C-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives aregenerally reacting the PEG polymer with a group that is reactive withhydroxyl groups, typically anhydrides, acid chlorides, chloroformates,and carbonates. In the second generation PEGylation chemistry moreefficient functional groups, such as aldehyde, esters, amides, etc., aremade available for conjugation.

As applications of PEGylation have become more and more advanced andsophisticated, there has been an increase in need for heterobifunctionalPEGs for conjugation. These heterobifunctional PEGs are very useful inlinking two entities, where a hydrophilic, flexible, and biocompatiblespacer is needed. Preferred end groups for heterobifunctional PEGs aremaleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids,and NHS esters.

The most common modification agents, or linkers, are based on methoxyPEG (mPEG) molecules. Their activity depends on adding aprotein-modifying group to the alcohol end. In some instancespolyethylene glycol (PEG diol) is used as the precursor molecule. Thediol is subsequently modified at both ends in order to make a hetero- orhomo-dimeric PEG-linked molecule.

Proteins are generally PEGylated at nucleophilic sites, such asunprotonated thiols (cysteinyl residues) or amino groups. Examples ofcysteinyl-specific modification reagents include PEG maleimide, PEGiodoacetate, PEG thiols, and PEG vinylsulfone. All four are stronglycysteinyl-specific under mild conditions and neutral to slightlyalkaline pH but each has some drawbacks. The thioether formed with themaleimides can be somewhat unstable under alkaline conditions so theremay be some limitation to formulation options with this linker. Thecarbamothioate linkage formed with iodo PEGs is more stable, but freeiodine can modify tyrosine residues under some conditions. PEG thiolsform disulfide bonds with protein thiols, but this linkage can also beunstable under alkaline conditions. PEG-vinylsulfone reactivity isrelatively slow compared to maleimide and iodo PEG; however, thethioether linkage formed is quite stable. Its slower reaction rate alsocan make the PEG-vinylsulfone reaction easier to control.

Site-specific PEGylation at native cysteinyl residues is seldom carriedout, since these residues are usually in the form of disulfide bonds orare required for biological activity. On the other hand, site-directedmutagenesis can be used to incorporate cysteinyl PEGylation sites forthiol-specific linkers. The cysteine mutation must be designed such thatit is accessible to the PEGylation reagent and is still biologicallyactive after PEGylation.

Amine-specific modification agents include PEG NHS ester, PEG tresylate,PEG aldehyde, PEG isothiocyanate, and several others. All react undermild conditions and are very specific for amino groups. The PEG NHSester is probably one of the more reactive agents; however, its highreactivity can make the PEGylation reaction difficult to control on alarge scale. PEG aldehyde forms an imine with the amino group, which isthen reduced to a secondary amine with sodium cyanoborohydride. Unlikesodium borohydride, sodium cyanoborohydride will not reduce disulfidebonds. However, this chemical is highly toxic and must be handledcautiously, particularly at lower pH where it becomes volatile.

Due to the multiple lysine residues on most proteins, site-specificPEGylation can be a challenge. Fortunately, because these reagents reactwith unprotonated amino groups, it is possible to direct the PEGylationto lower-pK amino groups by performing the reaction at a lower pH.Generally the pK of the alpha-amino group is 1-2 pH units lower than theepsilon-amino group of lysine residues. By PEGylating the molecule at pH7 or below, high selectivity for the N-terminus frequently can beattained. However, this is only feasible if the N-terminal portion ofthe protein is not required for biological activity. Still, thepharmacokinetic benefits from PEGylation frequently outweigh asignificant loss of in vitro bioactivity, resulting in a product withmuch greater in vivo bioactivity regardless of PEGylation chemistry.

There are several parameters to consider when developing a PEGylationprocedure. Fortunately, there are usually no more than four or five keyparameters. The “design of experiments” approach to optimization ofPEGylation conditions can be very useful. For thiol-specific PEGylationreactions, parameters to consider include: protein concentration,PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time,and in some instances, the exclusion of oxygen. (Oxygen can contributeto intermolecular disulfide formation by the protein, which will reducethe yield of the PEGylated product.) The same factors should beconsidered (with the exception of oxygen) for amine-specificmodification except that pH may be even more critical, particularly whentargeting the N-terminal amino group.

For both amine- and thiol-specific modifications, the reactionconditions may affect the stability of the protein. This may limit thetemperature, protein concentration, and pH. In addition, the reactivityof the PEG linker should be known before starting the PEGylationreaction. For example, if the PEGylation agent is only 70 percentactive, the amount of PEG used should ensure that only active PEGmolecules are counted in the protein-to-PEG reaction stoichiometry.

VI. PROTEINS AND PEPTIDES

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide, such as anengineered cyst(e)inease. These peptides may be comprised in a fusionprotein or conjugated to an agent as described supra.

As used herein, a protein or peptide generally refers, but is notlimited to, a protein of greater than about 200 amino acids, up to afull length sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. For convenience, the terms “protein,” “polypeptide,”and “peptide” are used interchangeably herein.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative, or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acids interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid.

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides, orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide, andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases (available onthe world wide web at ncbi.nlm.nih.gov/). The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins,polypeptides, and peptides are known to those of skill in the art.

VII. NUCLEIC ACIDS AND VECTORS

In certain aspects of the invention, nucleic acid sequences encoding anengineered cyst(e)inease or a fusion protein containing a modifiedcyst(e)inease may be disclosed. Depending on which expression system isused, nucleic acid sequences can be selected based on conventionalmethods. For example, if the engineered cyst(e)inease is derived fromprimate CGL and contains multiple codons that are rarely utilized in E.coli, then that may interfere with expression. Therefore, the respectivegenes or variants thereof may be codon optimized for E. coli expression.Various vectors may be also used to express the protein of interest,such as engineered cyst(e)inease. Exemplary vectors include, but are notlimited, plasmid vectors, viral vectors, transposon, or liposome-basedvectors.

VIII. HOST CELLS

Host cells may be any that may be transformed to allow the expressionand secretion of engineered cyst(e)inease and conjugates thereof. Thehost cells may be bacteria, mammalian cells, yeast, or filamentousfungi. Various bacteria include Escherichia and Bacillus. Yeastsbelonging to the genera Saccharomyces, Kiuyveromyces, Hansenula, orPichia would find use as an appropriate host cell. Various species offilamentous fungi may be used as expression hosts, including thefollowing genera: Aspergillus, Trichoderma, Neurospora, Penicillium,Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, andPyricularia.

Examples of usable host organisms include bacteria, e.g., Escherichiacoli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al.,1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcuslividans (Hopwood et al., 1985); yeasts, e.g., Saccharomyces cerevisiaeAH 22 (Mellor et al., 1983) or Schizosaccharomyces pombe; andfilamentous fungi, e.g., Aspergillus nidulans, Aspergillus awamori(Ward, 1989), or Trichoderma reesei (Penttila et al., 1987; Harkki etal., 1989).

Examples of mammalian host cells include Chinese hamster ovary cells(CHO-K1; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCCCRL 1548),SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and murineembryonic cells (NIH-3T3; ATCC CRL 1658). The foregoing beingillustrative but not limitative of the many possible host organismsknown in the art. In principle, all hosts capable of secretion can beused whether prokaryotic or eukaryotic.

Mammalian host cells expressing the engineered cyst(e)inease and/ortheir fusion proteins are cultured under conditions typically employedto culture the parental cell line. Generally, cells are cultured in astandard medium containing physiological salts and nutrients, such asstandard RPMI, MEM, IMEM, or DMEM, typically supplemented with 5%-10%serum, such as fetal bovine serum. Culture conditions are also standard,e.g., cultures are incubated at 37° C. in stationary or roller culturesuntil desired levels of the proteins are achieved.

IX. PROTEIN PURIFICATION

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue, or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity) unless otherwise specified. Analytical methods particularlysuited to the preparation of a pure peptide are ion-exchangechromatography, gel exclusion chromatography, polyacrylamide gelelectrophoresis, affinity chromatography, immunoaffinity chromatography,and isoelectric focusing. A particularly efficient method of purifyingpeptides is fast-performance liquid chromatography (FPLC) or evenhigh-performance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by centrifugation; chromatography steps,such as ion exchange, gel filtration, reverse phase, hydroxyapatite, andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

There is no general requirement that the protein or peptide will alwaysbe provided in its most purified state. Indeed, it is contemplated thatless substantially purified products may have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

In certain embodiments a protein or peptide may be isolated or purified,for example, an engineered cyst(e)inease, a fusion protein containingthe engineered AAD, or an engineered cyst(e)inease post PEGylation. Forexample, a His tag or an affinity epitope may be comprised in such anengineered cyst(e)inease to facilitate purification. Affinitychromatography is a chromatographic procedure that relies on thespecific affinity between a substance to be isolated and a molecule towhich it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that does not adsorb molecules to anysignificant extent and that has a broad range of chemical, physical, andthermal stability. The ligand should be coupled in such a way as to notaffect its binding properties. The ligand should also provide relativelytight binding. It should be possible to elute the substance withoutdestroying the sample or the ligand.

Size exclusion chromatography (SEC) is a chromatographic method in whichmolecules in solution are separated based on their size, or in moretechnical terms, their hydrodynamic volume. It is usually applied tolarge molecules or macromolecular complexes, such as proteins andindustrial polymers. Typically, when an aqueous solution is used totransport the sample through the column, the technique is known as gelfiltration chromatography, versus the name gel permeationchromatography, which is used when an organic solvent is used as amobile phase.

The underlying principle of SEC is that particles of different sizeswill elute (filter) through a stationary phase at different rates. Thisresults in the separation of a solution of particles based on size.Provided that all the particles are loaded simultaneously or nearsimultaneously, particles of the same size should elute together. Eachsize exclusion column has a range of molecular weights that can beseparated. The exclusion limit defines the molecular weight at the upperend of this range and is where molecules are too large to be trapped inthe stationary phase. The permeation limit defines the molecular weightat the lower end of the range of separation and is where molecules of asmall enough size can penetrate into the pores of the stationary phasecompletely and all molecules below this molecular mass are so small thatthey elute as a single band.

High-performance liquid chromatography (or high-pressure liquidchromatography, HPLC) is a form of column chromatography used frequentlyin biochemistry and analytical chemistry to separate, identify, andquantify compounds. HPLC utilizes a column that holds chromatographicpacking material (stationary phase), a pump that moves the mobilephase(s) through the column, and a detector that shows the retentiontimes of the molecules. Retention time varies depending on theinteractions between the stationary phase, the molecules being analyzed,and the solvent(s) used.

X. PHARMACEUTICAL COMPOSITIONS

It is contemplated that the novel cyst(e)inease can be administeredsystemically or locally to inhibit tumor cell growth and, mostpreferably, to kill cancer cells in cancer patients with locallyadvanced or metastatic cancers. They can be administered intravenously,intrathecally, and/or intraperitoneally. They can be administered aloneor in combination with anti-proliferative drugs. In one embodiment, theyare administered to reduce the cancer load in the patient prior tosurgery or other procedures. Alternatively, they can be administeredafter surgery to ensure that any remaining cancer (e.g., cancer that thesurgery failed to eliminate) does not survive.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided in formulations together withphysiologically tolerable liquid, gel, or solid carriers, diluents, andexcipients. These therapeutic preparations can be administered tomammals for veterinary use, such as with domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual subjects.

Such compositions are typically prepared as liquid solutions orsuspensions, as injectables. Suitable diluents and excipients are, forexample, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired, the compositions maycontain minor amounts of auxiliary substances, such as wetting oremulsifying agents, stabilizing agents, or pH buffering agents.

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions comprising proteins, antibodies, anddrugs in a form appropriate for the intended application. Generally,pharmaceutical compositions may comprise an effective amount of one ormore CGL variant or additional agents dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic, or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one CGL variant isolated by the method disclosedherein, or additional active ingredient will be known to those of skillin the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated hereinby reference. Moreover, for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by the FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed., 1990, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the pharmaceutical compositions is contemplated.

Certain embodiments of the present invention may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid, or aerosol form, and whether it needs to be sterile forthe route of administration, such as injection. The compositions can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, intramuscularly, subcutaneously, mucosally, orally,topically, locally, by inhalation (e.g., aerosol inhalation), byinjection, by infusion, by continuous infusion, by localized perfusionbathing target cells directly, via a catheter, via a lavage, in lipidcompositions (e.g., liposomes), or by other methods or any combinationof the forgoing as would be known to one of ordinary skill in the art(see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990,incorporated herein by reference).

The modified polypeptides may be formulated into a composition in a freebase, neutral, or salt form. Pharmaceutically acceptable salts includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganicacids, such as, for example, hydrochloric or phosphoric acids, or suchorganic acids as acetic, oxalic, tartaric, or mandelic acid. Saltsformed with the free carboxyl groups can also be derived from inorganicbases, such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine, or procaine. Upon formulation, solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective. The formulations areeasily administered in a variety of dosage forms, such as formulated forparenteral administrations, such as injectable solutions, or aerosolsfor delivery to the lungs, or formulated for alimentary administrations,such as drug release capsules and the like.

Further in accordance with certain aspects of the present invention, thecomposition suitable for administration may be provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent, or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a composition contained therein, its use inadministrable composition for use in practicing the methods isappropriate. Examples of carriers or diluents include fats, oils, water,saline solutions, lipids, liposomes, resins, binders, fillers, and thelike, or combinations thereof. The composition may also comprise variousantioxidants to retard oxidation of one or more component. Additionally,the prevention of the action of microorganisms can be brought about bypreservatives, such as various antibacterial and antifungal agents,including but not limited to parabens (e.g., methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof.

In accordance with certain aspects of the present invention, thecomposition is combined with the carrier in any convenient and practicalmanner, i.e., by solution, suspension, emulsification, admixture,encapsulation, absorption, and the like. Such procedures are routine forthose skilled in the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner, such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in acomposition include buffers, amino acids, such as glycine and lysine,carbohydrates, such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle composition that includes CGL variants, oneor more lipids, and an aqueous solvent. As used herein, the term “lipid”will be defined to include any of a broad range of substances that ischaracteristically insoluble in water and extractable with an organicsolvent. This broad class of compounds is well known to those of skillin the art, and as the term “lipid” is used herein, it is not limited toany particular structure. Examples include compounds that containlong-chain aliphatic hydrocarbons and their derivatives. A lipid may benaturally occurring or synthetic (i.e., designed or produced by man).However, a lipid is usually a biological substance. Biological lipidsare well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether- andester-linked fatty acids, polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the engineered cyst(e)inease or a fusion proteinthereof may be dispersed in a solution containing a lipid, dissolvedwith a lipid, emulsified with a lipid, mixed with a lipid, combined witha lipid, covalently bonded to a lipid, contained as a suspension in alipid, contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition administered to an animalpatient can be determined by physical and physiological factors, such asbody weight, severity of condition, the type of disease being treated,previous or concurrent therapeutic interventions, idiopathy of thepatient, and on the route of administration. Depending upon the dosageand the route of administration, the number of administrations of apreferred dosage and/or an effective amount may vary according to theresponse of the subject. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared in such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors, such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations, will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 milligram/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 milligram/kg/body weightto about 100 milligram/kg/body weight, about 5 microgram/kg/body weightto about 500 milligram/kg/body weight, etc., can be administered, basedon the numbers described above.

XI. COMBINATION TREATMENTS

In certain embodiments, the compositions and methods of the presentembodiments involve administration of a cyst(e)inease in combinationwith a second or additional therapy. Such therapy can be applied in thetreatment of any disease that is associated with cyst(e)ine dependency.For example, the disease may be cancer.

The methods and compositions, including combination therapies, enhancethe therapeutic or protective effect, and/or increase the therapeuticeffect of another anti-cancer or anti-hyperproliferative therapy.Therapeutic and prophylactic methods and compositions can be provided ina combined amount effective to achieve the desired effect, such as thekilling of a cancer cell and/or the inhibition of cellularhyperproliferation. This process may involve administering to the cellsboth a cyst(e)inease and a second therapy. A tissue, tumor, or cell canbe exposed to one or more compositions or pharmacological formulation(s)comprising one or more of the agents (i.e., a cyst(e)inease or ananti-cancer agent), or by contacting the tissue, tumor, and/or cell withtwo or more distinct compositions or formulations, wherein onecomposition provides 1) a cyst(e)inease, 2) an anti-cancer agent, or 3)both a cyst(e)inease and an anti-cancer agent. Also, it is contemplatedthat such a combination therapy can be used in conjunction withchemotherapy, radiotherapy, surgical therapy, or immunotherapy.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing, for example, both agents are delivered to a cellin a combined amount effective to kill the cell or prevent it fromdividing.

A cyst(e)inease may be administered before, during, after, or in variouscombinations relative to an anti-cancer treatment. The administrationsmay be in intervals ranging from concurrently to minutes to days toweeks. In embodiments where the cyst(e)inease is provided to a patientseparately from an anti-cancer agent, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the two compounds would still be able to exert anadvantageously combined effect on the patient. In such instances, it iscontemplated that one may provide a patient with the cyst(e)inease andthe anti-cancer therapy within about 12 to 24 or 72 h of each other and,more particularly, within about 6-12 h of each other. In some situationsit may be desirable to extend the time period for treatmentsignificantly where several days (2, 3, 4, 5, 6, or 7) to several weeks(1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

In certain embodiments, a course of treatment will last 1-90 days ormore (this such range includes intervening days). It is contemplatedthat one agent may be given on any day of day 1 to day 90 (this suchrange includes intervening days) or any combination thereof, and anotheragent is given on any day of day 1 to day 90 (this such range includesintervening days) or any combination thereof. Within a single day(24-hour period), the patient may be given one or multipleadministrations of the agent(s). Moreover, after a course of treatment,it is contemplated that there is a period of time at which noanti-cancer treatment is administered. This time period may last 1-7days, and/or 1-5 weeks, and/or 1-12 months or more (this such rangeincludes intervening days), depending on the condition of the patient,such as their prognosis, strength, health, etc. It is expected that thetreatment cycles would be repeated as necessary.

Various combinations may be employed. For the example below acyst(e)inease is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

A. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present embodiments. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gamma1I andcalicheamicin omegaI1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine,navelbine, farnesyl-protein transferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove.

B. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

C. Immunotherapy

The skilled artisan will understand that immunotherapies may be used incombination or in conjunction with methods of the embodiments. In thecontext of cancer treatment, immunotherapeutics, generally, rely on theuse of immune effector cells and molecules to target and destroy cancercells. Rituximab (RITUXAN®) is such an example. The immune effector maybe, for example, an antibody specific for some marker on the surface ofa tumor cell. The antibody alone may serve as an effector of therapy orit may recruit other cells to actually affect cell killing. The antibodyalso may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present embodiments. Common tumormarkers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,erb B, and p155. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines, such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growthfactors, such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998);cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998);gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998;Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-gangliosideGM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat.No. 5,824,311). It is contemplated that one or more anti-cancertherapies may be employed with the antibody therapies described herein.

D. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present embodiments, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

E. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

XII. KITS

Certain aspects of the present invention may provide kits, such astherapeutic kits. For example, a kit may comprise one or morepharmaceutical composition as described herein and optionallyinstructions for their use. Kits may also comprise one or more devicesfor accomplishing administration of such compositions. For example, asubject kit may comprise a pharmaceutical composition and catheter foraccomplishing direct intravenous injection of the composition into acancerous tumor. In other embodiments, a subject kit may comprisepre-filled ampoules of an engineered cyst(e)inease, optionallyformulated as a pharmaceutical, or lyophilized, for use with a deliverydevice.

Kits may comprise a container with a label. Suitable containers include,for example, bottles, vials, and test tubes. The containers may beformed from a variety of materials, such as glass or plastic. Thecontainer may hold a composition that includes an engineeredcyst(e)inease that is effective for therapeutic or non-therapeuticapplications, such as described above. The label on the container mayindicate that the composition is used for a specific therapy ornon-therapeutic application, and may also indicate directions for eitherin vivo or in vitro use, such as those described above. The kit of theinvention will typically comprise the container described above and oneor more other containers comprising materials desirable from acommercial and user standpoint, including buffers, diluents, filters,needles, syringes, and package inserts with instructions for use.

XIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Cystathionine-γ-Lyase as a Scaffold for a Human Cyst(e)IneaseEnzyme

Due to the undesired effects of immunogenicity seen clinically with theuse of non-human protein therapeutics, the inventors sought to engineertherapeutically relevant cystine/cysteine degrading activity into ahuman enzyme (i.e., engineer an enzyme with high k_(cat) and low K_(M)values for cystine/cysteine and also displaying a favorablespecificity). Humans have an enzyme called cystathionine-γ-lyase (hCGL)whose function is to catalyze the last step in the mammaliantranssulfuration pathway (Rao et al., 1990), namely the conversion ofL-cystathionine to L-cysteine, alpha-ketobutyrate, and ammonia. HumanCGL can also weakly degrade L-cysteine and its disulfide form,L-cystine, making it an ideal candidate for engineering. Usingstructurally- and phylogenetically-guided mutagenesis, hCGL variantswere engineered to efficiently hydrolyze both L-cysteine and L-cystine.

Example 2—Gene Synthesis and Expression of Human Cysthionine-γ-Lyase andHuman Cyst(e)Inease

The hCGL gene contains multiple codons that are rarely utilized in E.coli and can interfere with expression. Thus, in order to optimizeprotein expression in E. coli, the respective genes were assembled withcodon optimized oligonucleotides designed using the DNA-Works software(Hoover et al., 2002). Each construct contains an N-terminal NcoIrestriction site, an in-frame N-terminal His₆ tag and a C-terminal EcoRIsite for simplifying cloning. After cloning into a pET28a vector(Novagen), E. coli (BL21) containing an appropriate hCGL expressionvector were grown at 37° C. using Terrific Broth (TB) media containing50 μg/ml kanamycin in shaking flasks at 250 rpm until reaching an OD₆₀₀of ˜0.5-0.6. At this point the cultures were switched to a shaker at 25°C. and induced with 0.5 mM IPTG and allowed to express protein for anadditional 12 h. Cell pellets were then collected by centrifugation andre-suspended in an IMAC buffer (10 mM NaPO₄/10 mM imidazole/300 mM NaCl,pH 8). After lysis by a French pressure cell, lysates were centrifugedat 20,000×g for 20 min at 4° C., and the resulting supernatant appliedto a nickel IMAC column, washed with 10-20 column volumes of IMACbuffer, and then eluted with an IMAC elution buffer (50 mM NaPO₄/250 mMimidazole/300 mM NaCl, pH 8). Fractions containing enzyme were thenincubated with 10 mM pyridoxal phosphate (PLP) for an hour at 25° C.Using a 10,000 MWCO centrifugal filter device (AMICON™), proteins werethen buffer exchanged several times into a 100 mM PBS, 10% glycerol, pH7.3 solution. Aliquots of hCGL enzyme or hCGL-variant enzyme were thenflash frozen in liquid nitrogen and stored at −80° C. hCGL orhCGL-variant enzyme purified in this manner was >95% homogeneous asassessed by SDS-PAGE and coomassie staining. The yield was calculated tobe ˜200-300 mg/L culture based upon the calculated extinctioncoefficient, ε₂₈₀=29,870 M⁻¹ cm⁻¹ in a final buffer concentration of 6 Mguanidinium hydrochloride, 20 mM phosphate buffer, pH 6.5 (Gill and vonHippel, 1989).

Example 3—96-Well Plate Screen for Cyst(e)Inease Activity and RankingClones

Human CGL slowly degrades L-cysteine to pyruvate, ammonia and H₂S, andconverts L-cystine to pyruvate, ammonia and thiocysteine (k_(cat)/K_(M)˜0.2 s⁻¹ mM⁻¹ and 0.5 s⁻¹ mM⁻¹, respectively) (FIG. 1). Thiocysteine isfurther nonenzymatically degraded to L-cysteine and H₂S. A colorimetricassay for the detection of pyruvate using 3-methyl-2-benzothiazolinonehydrazone (MBTH) (Takakura et al., 2004) was scaled to a 96-well plateformat for screening small libraries and for ranking clones with thegreatest cystine and/or cysteine lyase activity. This plate screenprovides a facile method for picking the most active clones from themutagenic libraries. Clones displaying greater activity than parentalcontrols were selected for further characterization.

Single colonies containing mutagenized hCGL, or hCGL controls, werepicked into 96-well culture plates containing 75 μL of TB media/wellcontaining 50 μg/ml kanamycin. These cultures are then grown at 37° C.on a plate shaker until reaching an OD₆₀₀ of ˜0.8-1. After cooling to25° C., an additional 75 μL of media/well containing 50 μg/ml kanamycinand 2 mM IPTG was added. Expression was performed at 25° C. with shakingfor at least 2 h, following which 100 μL of culture/well was transferredto a 96-well assay plate. The assay plates were then centrifuged topellet the cells, the media was removed, and the cells were lysed byaddition of 50 μL/well of B-PER protein extraction reagent (Pierce).After clearing by centrifugation the lysate was split between two platesfor incubation with 0.8 mM L-cystine in one plate and 1.2 mM L-cysteinein the other and incubated 37° C. for 10-12 hrs. The reaction was thenderivatized by addition of 3 parts of 0.03% MBTH solution in 1 M sodiumacetate pH 5. The plates were heated at 50° C. for 40 min and aftercooling were read at λ=320 nm in a microtiter plate reader.

Example 4—Effect of Mutagenesis Upon Residues E59, R119, and E339 ofhCGL

Structural analysis indicated that residues E59, R119, and E339 werelikely involved in the recognition of hCGL for it substrateL-cystathionine. NNS codon (N can be A, T, G, or C; S can be G or C)saturation libraries were constructed at these sites and screened usingthe following mutagenic primers: (E59) Forward5′-GGCCAGCATAGCGGTTTTNNSTATAGCCGTAGCGGC (SEQ ID NO: 12), Reverse5′-GCCGCTACGGCTATASNNAAAACCGCTATGCTGGCC (SEQ ID NO: 13), (R119) Forward5′-GTATGGTGGGACCAATNNSTATTTCCGTCAGGTGGCG (SEQ ID NO: 14), Reverse5′-CGCCACCTGACGGAAATASNNATTGGTCCCACCATAC (SEQ ID NO: 15), (E339) Forward5′-CTGAAACTGTTTACCCTGGCANNSAGCTTGGGCGGCTTTG (SEQ ID NO: 16), and Reverse5′-CAAAGCCGCCCAAGCTSNNTGCCAGGGTAAACAGTTTCAG (SEQ ID NO: 17), using thehCGL gene as template DNA and specific end primers; forward5′-GATATACCATGGGAGGCCATCACCACCATCATCATGGCGGGCAGGAAAAGGATGCG (Seq ID NO:18) and reverse5′-CTCGAATTCTCAACTGTGGCTTCCCGATGGGGGATGGGCCGCTTTCAGCGCCTGATCC (SEQ IDNO: 19). The PCR product was digested with NcoI and EcoRI and ligatedinto pET28a vector with T4 DNA ligase. The resulting ligations weretransformed directly into E. coli (BL21) and plated on LB-kanamycinplates for subsequent screening as described in Example 3. All librarieswere screened in two-fold excess of their theoretical size. Clonesdisplaying activity were isolated and the sequences of the hCGL genevariants were determined to identify the mutations.

The enzyme variants were purified to greater than 95% homogeneity asassessed by SDS-PAGE. Incubation with PLP was shown to enhance thespecific activity presumably because the E. coli cells used forexpression do not produce sufficient PLP for the amount of enzymeproduced. Once the enzyme had been loaded with PLP it was stable and noloss of the cofactor and thus no decrease in activity could be detectedfollowing several days of storage.

Example 5—Characterization of Human Cyst(e)Inease Variant hCGL-TV

One particular variant identified from the screen as having the highestcatalytic activity for degrading both L-cystine and L-cysteine was foundto have the following mutations: E59T, a synonymous codon change ofR119R, and E339V. This variant was called hCGL-TV (FIG. 5) and wascharacterized for its ability to degrade L-cyst(e)ine in a 100 mM PBSbuffer at pH 7.3 and 37° C. using a 1 mL scale MBTH assay similar tothat described in Example 3. Under these conditions, the hCGL-TV variantwas found to degrade L-cystine with a k_(cat) of 1.0±0.05 s⁻¹, a K_(M)of 0.16±0.02 mM, and a k_(cat)/K_(M) of 6.3±1.0 s⁻¹ mM⁻¹ (FIG. 2A). ThehCGL-TV variant was further found to have a k_(cat) of 0.8±0.03 s⁻¹, aK_(M) of 0.25±0.04 mM, and a k_(cat)/K_(M) of 3.2±0.6 s⁻¹ mM⁻¹ fordegradation of L-cysteine (FIG. 2B).

The serum stability of hCGL-TV was tested by incubation in pooled humanserum at 37° C. At time points, aliquots were withdrawn and tested forremaining activity using L-cystine as a substrate. After plotting thedata, the hCGL-TV variant was found to be very stable with an apparentT_(0.5) of 228±6 h (FIG. 3).

Example 6—In Vitro Cytotoxicity of hCGL-TV Against Prostate Tumor CellLines

The in vitro cytotoxicity of hCGL-TV was assessed using DU145 and PC3prostate tumor cell lines. Cells were seeded at ˜3000 cells/well inRPMI-1640 media and allowed to grow for 24 h before titrations ofhCGL-TV ranging from 0-10 μM were added to each well. After incubationfor 3 days the relative number of surviving cells was assessedcolorimetrically using the(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay. Analysis of the resulting data yielded an apparent IC₅₀value of ˜60 nM for both the DU145 cell line and the PC3 cell line(FIGS. 4A and 4B, respectively).

Example 7—Pharmacological Preparation of Human Cyst(e)Inease

The hCGL-TV enzyme was purified as described in Example 2 with oneexception: after binding to the IMAC column, the protein is washedextensively (90-100 column volumes) with an IMAC buffer containing 0.1%TRITON™ 114 in the sample. The sample was washed again with 10-20 columnvolumes of IMAC buffer, and then eluted with an IMAC elution buffer (50mM NaPO₄/250 mM imidazole/300 mM NaCl, pH 8). The wash with TRITON™ 114was employed for endotoxin removal. The purified protein was subjectedto buffer exchange into a 100 mM NaPO₄ buffer at pH 8.3 using a 10,000MWCO filtration device (AMICON®). Subsequently, PLP was added at aconcentration of 10 mM and the protein was incubated for 1 h at 25° C.Methoxy PEG Succinimidyl Carboxymethyl Ester 5000 MW (JenKem Technology)was then added to hCGL-TV at an 80:1 molar ratio and allowed to reactfor 1 h at 25° C. under constant stirring. The resulting mixture wasextensively buffer exchanged (PBS with 10% glycerol) using a 100,000MWCO filtration device (AMICON®), and sterilized with a 0.2 micronsyringe filter (VWR). All PEGylated enzymes were analyzed forlipopolysaccharide (LPS) content using a Limulus Amebocyte Lysate (LAL)kit (Cape Cod Incorporated).

Example 8—Engineering of Primate Cyst(e)Ineases

The sequences of CGLs from primate species, such as chimpanzees (Pantroglodytes; SEQ ID NO: 9), bonobos (Pan paniscus; SEQ ID NO: 10),orangutans (Pongo abelii; SEQ ID NO: 7), and macaques (Macacafascicularis; SEQ ID NO: 8) are, respectively, about 99.3%, 98.8%, 96%,and 95.3% identical in amino acid composition to human CGL (SEQ ID NO:1). Due to the sequence similarity, engineered CGLs from these organismswill not likely cause significant immune responses if introduced intohumans as a therapeutic. Primate CGL enzymes with mutations conferringenhanced cyst(e)inease activity will be constructed using standardmutagenesis techniques as described in Example 4. The resulting geneswill be cloned into pET28a and the sequence verified to ensure that noundesired mutations are incorporated. The constructs used to express andpurify the resulting enzymes are described above. Primate CGLsengineered with amino acid positions corresponding to 59T and 339V (SEQID NOs: 3-6) are expected to have enhanced activity for both L-cystineand L-cysteine with k_(cat)/K_(M) values of at least 1×10³ s⁻¹M⁻¹ orhigher. The serum stability at 37° C. of the engineered primate CGLswith enhanced cyst(e)inease activity will be determined as describedabove.

Example 9—Pharmacodynamic (PD) Analyses of PEG-hCGL-TV in Mice

Male FVB mice were injected i.p. with 50 mg/kg of PEG-hCGL-TV andsacrificed at days 0, 1, 2, 4, and 6 (n=5 per group) for blood and serumcollection. Serum samples were mixed with an internal standard mixtureof 10 picomole deuterated cystine and cysteine and ultrafiltered usingNANOSEP® OMEGA™ centrifugal devices, 3 kDa cutoff (Pall LifeBiosciences) (Tiziani et al., 2008; Tiziani et al., 2013). The filteredpolar fractions were chromatographed using a reverse-phase BEH C18, 1.7μm, 2.1×150 mm column (THERMO SCIENTIFIC™ ACCELA® 1250 UPLC, WatersCorporation, USA) and introduced into an EXACTIVE™ Plus ORBITRAP™ massspectrometer coupled with electrospray ionization (Thermo FisherScientific, San Jose, Calif.). Data was acquired in centroid MS modefrom 50 to 700 m/z mass range with the XCALIBUR™ software provided withinstrument. The relative concentrations of cystine and cysteine arereported as mean values±SEM. As can be seen in FIG. 6A, PEG-hCGL-TVdrastically reduces serum cystine levels (>95%) for over 96 h, and an80% reduction in cysteine levels for over 48 h (FIG. 6B).

Example 10—Pharmacokinetic (PK) Analyses of PEG-hCGL-TV in Mice

The circulatory persistence of PEG-hCGL-TV was assessed using the sameserum samples as described in Example 9. Using a dot blot densitometrytechnique, samples were probed with an anti-hCGL antibody (rabbitanti-CTH Sigma # C8248) followed by addition of anti-rabbit IgG-FITC(Santa Cruz Biotechnology # sc-2012) for visualization by excitation at488 nm on a TYPHOON™ scanner (GE Healthcare). Using ImageJ software(Schneider et al., 2012), densitometry bands of the samples werecompared to titrations of known amounts of PEG-hCGL-TV within the sameblot to construct a standard curve and calculate relative serumPEG-hCGL-TV levels. Fitting this data to an extravascular model ofadministration (Foye et al., 2007; Stone et al., 2012) demonstrated anabsorption T_(1/2) of approximately 23 h, and an elimination T_(1/2) of40±7 h for PEG-hCGL-TV (FIG. 7).

Example 11—Effect of PEG-hCGL-TV on the Growth of HMVP2 Tumor Cells inan Allograft Model

The ability of PEG-hCGL-TV to inhibit tumor development was evaluated ina mouse prostate carcinoma (HMVP2) allograft model. For this experiment,HMVP2 cells were grown as spheroids and used to initiate tumors in theflanks of four groups (eight mice/group) of FVB/N male mice followings.c. injection. Cells were grown for approximately two weeks, at whichtime small tumors at the injection site became palpable. Each of thefour groups was then administered either PEG-hCGL-TV at 50 or 100 mg/kg,PBS (control), or heat-inactivated PEG-hCGL-TV (additional control) at100 mg/kg by i.p. injection every 4 days. As shown in FIG. 8, treatmentwith PEG-hCGL-TV produced a dramatic inhibition of tumor growth, whichwas highly statistically significant at both doses (50 and 100 mg/kg)compared to both control groups (i.e., PBS injection alone andinactivated enzyme injection control groups). Importantly, repeateddosing was very well tolerated throughout the treatment period. In thisregard, there were no differences in body weight or food consumptionamong the treated and control groups of mice over the course of theexperiment. Finally, at the termination of this experiment, mice werenecropsied and no abnormalities were seen in any major organs uponmacroscopic evaluation.

Example 12—Effect of PEG-hCGL-TV on the Growth of PC3 Prostate TumorCells in a Xenograft Model

The ability of PEG-hCGL-TV to inhibit tumor development was evaluated ina human prostate carcinoma (PC3) xenograft model. For this experiment,PC3 cells were used to initiate tumors in the flanks of four groups(seven mice/group) of FVB/N male mice following s.c. injection. Cellswere grown for 10 days at which time tumors at the injection site becamepalpable. Each of the four groups was then administered eitherPEG-hCGL-TV at 50 or 100 mg/kg, PBS (control), or heat-inactivatedPEG-hCGL-TV (additional control) at 100 mg/kg by i.p. injection every 4days. As can be seen in FIG. 9, treatment with PEG-hCGL-TV caused apronounced retardation of tumor growth, at both doses (50 and 100 mg/kg)compared to both control groups (i.e., PBS injection alone andinactivated enzyme injection control groups). Repeated dosing was alsovery well tolerated in this mouse strain throughout the treatment periodwith no differences in body weight or food consumption among the treatedand control groups of mice over the course of the experiment. At thetermination of this experiment, mice were necropsied and noabnormalities were seen in any major organs upon macroscopic evaluation.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,870,287-   U.S. Pat. No. 5,739,169-   U.S. Pat. No. 5,760,395-   U.S. Pat. No. 5,801,005-   U.S. Pat. No. 5,824,311-   U.S. Pat. No. 5,830,880-   U.S. Pat. No. 5,846,945-   U.S. Pat. No. 5,889,155-   U.S. Pat. Publn. 2009/0304666-   Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845,    1998.-   Ausubel et al., Current Protocols in Molecular Biology, Greene    Publishing Associates and Wiley Interscience, N.Y., 1994.-   Bukowski et al., Clinical Cancer Res., 4(10):2337-2347, 1998.-   Christodoulides et al., Microbiology, 144(Pt 11):3027-3037, 1998.-   Davidson et al., J. Immunother., 21(5):389-398, 1998.-   Doxsee et al., Sulfasalazine-induced cystine starvation: Potential    use for prostate cancer therapy. The Prostate, 67(2):162-171, 2007.-   Foye et al., Foye's Principles of Medicinal Chemistry, Lippincott    Williams & Wilkins, 2007.-   Gill and von Hippel, Calculation of protein extinction coefficients    from amino acid sequence data. Anal Biochem, 182(2):319-326, 1989.-   Glode et al., Cysteine auxotrophy of human leukemic lymphoblasts is    associated with decreased amounts of intracellular cystathionase    protein. Biochemistry, 20(5):1306-1311, 1981.-   Guan et al., The x c-cystine/glutamate antiporter as a potential    therapeutic target for small-cell lung cancer: use of sulfasalazine.    Cancer Chemotherapy and Pharmacology, 64(3):463-472, 2009.-   Hanibuchi et al., Int. J. Cancer, 78(4):480-485, 1998.-   Harkki et al., BioTechnology, 7:596-603, 1989.-   Hellstrand et al., Acta Oncologica, 37(4):347-353, 1998.-   Hollander, Front. Immun., 3:3, 2012.-   Hopwood et al., In: Genetic Manipulation of Streptomyces, A    Laboratory Manual, The John Innes Foundation, Norwich, Conn., 1985.-   Hoover et al., The structure of human macrophage inflammatory    protein-3alpha/CCL20. Linking antimicrobial and CC chemokine    receptor-6-binding activities with human beta-defensins. J Biol    Chem, 277(40):37647-37654, 2002.-   Kim et al., Expression of cystathionine β-synthase is downregulated    in hepatocellular carcinoma and associated with poor prognosis.    Oncology Reports, 21(6):1449-1454, 2009.-   Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998.-   Ito et al., J. Biochem., 79:1263, 1976.-   Link et al., Cystathionase: a potential cytoplasmic marker of    hematopoietic differentiation. Blut, 47(1):31-39, 1983.-   Lordanescu, J. Bacteriol, 12:597 601, 1975.-   Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring    Harbor Press, Cold Spring Harbor, N.Y., 1988.-   Mellor et al., Gene, 24:1-14, 1983.-   Penttila et al., Gene, 61:155-164, 1987.-   Qin et al., Proc. Natl. Acad. Sci. USA, 95(24):14411-14416, 1998.-   Rao et al., Role of the Transsulfuration Pathway and of    {gamma}-Cystathionase Activity in the Formation of Cysteine and    Sulfate from Methionine in Rat Hepatocytes. Journal of Nutrition,    120(8):837, 1990.-   Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,    1289-1329, 1990.-   Schneider et al., 671 nih image to imageJ: 25 years of image    analysis. Nature Methods, 9:2012.-   Sibakov et al., Eur. J. Biochem., 145:567 572, 1984.-   Stone et al., Strategies for optimizing the serum persistence of    engineered human arginase I for cancer therapy. Journal of    Controlled Release, 158:171-179, 2012.-   Takakura et al., Assay method for antitumor L-methionine-lyase:    comprehensive kinetic analysis of the complex reaction with    L-methionine. Analytical Biochemistry, 327(2):233-240, 2004.-   Tiziani et al., Optimized metabolite extraction from blood serum for    1H nuclear magnetic resonance spectroscopy. Analytical Biochemistry,    377:16-23, 2008.-   Tiziani et al., Metabolomics of the tumor microenvironment in    pediatric acute lymphoblastic leukemia. PLoS One, 8:e82859, 2013.-   Ward, Proc, Embo-Alko Workshop on Molecular Biology of Filamentous    Fungi, Helsinki, 119-128, 1989.-   Wawrzynczak and Thorpe, In: Immunoconjugates, Antibody Conuugates In    Radioimaging And Therapy Of Cancer, Vogel (Ed.), NY, Oxford    University Press, 28, 1987.-   Zhang et al., Stromal control of cystine metabolism promotes cancer    cell survival in chronic lymphocytic leukaemia. Nature Cell Biology,    14(3):276-286, 2012.-   Zhao et al., Frequent Epigenetic Silencing of the    Folate-Metabolising Gene Cystathionine-Beta-Synthase in    Gastrointestinal Cancer. PLoS One, 7(11):e49683, 2012.

What is claimed is:
 1. An isolated, modified primatecystathionine-γ-lyase (CGL) enzyme having at least one substitutionrelative to a native primate CGL amino acid sequence (see SEQ ID NOs: 1and 7-10), said at least one substitution including a threonine atposition 59 of the native primate CGL sequence.
 2. The enzyme of claim1, further comprising a valine substitution at position
 339. 3. Theenzyme of claim 1, further comprising a heterologous peptide segment. 4.The enzyme of claim 3, wherein the heterologous peptide segment is anXTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide. 5.The enzyme of claim 1, wherein the enzyme is coupled to polyethyleneglycol (PEG).
 6. The enzyme of claim 5, wherein the enzyme is coupled toPEG via one or more lysine or cystine residues.
 7. An isolated, modifiedprimate cystathionine-γ-lyase (CGL) enzyme having at least twosubstitutions relative to a native primate CGL amino acid sequence (seeSEQ ID NOs: 1 and 7-10), said at least two substitutions including athreonine at position 59 and a valine at position 339 of the nativeprimate CGL sequence.
 8. A nucleic acid comprising a nucleotide sequenceencoding the enzyme of claim
 1. 9. The nucleic acid of claim 8, whereinthe nucleic acid is codon optimized for expression in bacteria, fungus,insects, or mammals.
 10. An expression vector comprising the nucleicacid of claim
 8. 11. A host cell comprising the nucleic acid of claim 8.12. The host cell of claim 11, wherein the host cell is a bacterialcell, a fungal cell, an insect cell, or a mammalian cell.